High sugar diet promotes tumor progression paradoxically through aberrant upregulation of pepck1

High dietary sugar (HDS), a contemporary dietary concern due to excessive intake of added sugars and carbohydrates, escalates the risk of metabolic disorders and concomitant cancers. However, the molecular mechanisms underlying HDS-induced cancer progression are not completely understood. We found that phosphoenolpyruvate carboxykinase 1 (PEPCK1), a pivotal enzyme in gluconeogenesis, is paradoxically upregulated in tumors by HDS, but not by normal dietary sugar (NDS), during tumor progression. Targeted knockdown of pepck1, but not pepck2, specifically in tumor tissue in Drosophila in vivo, not only attenuates HDS-induced tumor growth but also significantly improves the survival of Ras/Src tumor-bearing animals fed HDS. Interestingly, HP1a-mediated heterochromatin interacts directly with the pepck1 gene and downregulates pepck1 gene expression in wild-type Drosophila. Mechanistically, we demonstrated that, under HDS conditions, pepck1 knockdown reduces both wingless and TOR signaling, decreases evasion of apoptosis, reduces genome instability, and suppresses glucose uptake and trehalose levels in tumor cells in vivo. Moreover, rational pharmacological inhibition of PEPCK1, using hydrazinium sulfate, greatly improves the survival of tumor-bearing animals with pepck1 knockdown under HDS. This study is the first to show that elevated levels of dietary sugar induce aberrant upregulation of PEPCK1, which promotes tumor progression through altered cell signaling, evasion of apoptosis, genome instability, and reprogramming of carbohydrate metabolism. These findings contribute to our understanding of the complex relationship between diet and cancer at the molecular, cellular, and organismal levels and reveal PEPCK1 as a potential target for the prevention and treatment of cancers associated with metabolic disorders. Supplementary Information The online version contains supplementary material available at 10.1007/s00018-024-05438-2.


Introduction
High dietary sugar (HDS) is a contemporary health issue characterized by elevated consumption of total sugars, including both added and free sugars, which escalates the risk of metabolic disorders and their associated cancers [1].Indeed, diet is a key lifestyle factor that significantly contributes to increased cancer risk; however, the mechanisms remain unclear and await further investigation [2].Therefore, investigating the role of HDS-induced metabolic dysfunction in promoting cancer progression is essential for the prevention and treatment of diet-associated cancers.Across diverse animal models, HDS has been observed to enhance tumor growth and promote cancer progression through modifications in growth signaling and metabolic states within cancer cells [3][4][5].Under HDS conditions, cancer cells exhibit the capacity to adapt their growth signaling and metabolic states, potentially involving upregulation of the wingless/ Wnt pathway and mTOR signaling, along with evasion of apoptosis and genome instability, which contributes to elevated mutation rates [6][7][8][9].However, the precise molecular mechanisms underlying HDS-induced cancer progression via metabolic reprogramming remain unclear.

Cellular and Molecular Life Sciences
Yu-Hshun Chin and Meng-Syuan Liu have contributed equally to this work.
Insufficient provision of glucose often works against the increased nutritional requirements of tumors, highlighting the importance of gluconeogenesis, a critical glucose anabolism process for synthesis of glucose from noncarbohydrate precursors, in fueling tumor cell growth [10,11].Phosphoenolpyruvate carboxykinases (PEPCKs or PCKs), including PEPCK1 (cytosolic isoform) and PEPCK2 (mitochondrial isoform), catalyze the first rate-limiting step in the gluconeogenesis pathway and exhibit conserved functions across species [12].Research has underscored the role of PEPCKs in promoting cancer, particularly PEPCK1, which facilitates colon cancer growth by increasing gluconeogenesis metabolites through phosphoenolpyruvate and pyruvate production, along with mTOR signaling activation [10,13,14].Additionally, PEPCK1 inhibition has been shown to decrease colon cancer cell growth by downregulating lactate utilization [15].However, whether and how PEPCK1/2 regulate HDS-induced cancer progression remain completely unexplored.
In this study, we utilized Drosophila Ras/Src cancer models to explore the role of paradoxical pepck1/2 upregulation in HDS-induced tumor progression.Taking advantage of the genetic conservation between Drosophila melanogaster and humans, particularly in genes associated with disease, such as oncogenes and tumor suppressor genes, we investigated the impact of pepck1/2 upregulation in Ras/Src-induced tumor growth [16].Notably, evolutionary conservation of the Ras protein family underscores its pivotal role in regulating cellular growth, differentiation, and survival signaling, while C-terminal Src kinase (csk) functions as a crucial regulatory protein primarily involved in the negative regulation of Src family proteins [17].
Our prior studies have uncovered new epigenetic functions of heterochromatin formation, mediated by heterochromatin protein 1a (HP1a), in inhibiting HDS-induced tumor progression [18].However, the mechanisms by which HDSinduced tumor progression occurs, particularly through metabolic reprogramming, are not fully understood.Here, through an unbiased bioinformatics approach, we surprisingly identified pepck1 as a gene significantly upregulated in tumors when exposed to HDS, whereas such an upregulation does not occur with normal dietary sugar (NDS).We also demonstrated that PEPCK1 is a direct target of regulation via HP1a-mediated heterochromatin formation.Interestingly, we also found that pepck2 was upregulated, though to a lesser extent than pepck1, during HDS-induced tumor progression.To elucidate the role of these pivotal gluconeogenesis genes in HDS-induced tumor growth, we created ras G12V , pepck1 RNAi ; csk −/− and ras G12V , pepck2 RNAi ; csk −/− Drosophila models that carry Ras/Src-induced tumors with pepck1 and pepck2 RNAi knockdown, respectively.This approach effectively decreased the excessive pepck1 or pepck2 expression observed in ras G12V ; csk −/− tumor-bearing animals.Moreover, we also created Drosophila models that carry Ras/Src-induced tumors with pepck1 or pepck2 overexpression, respectively.Importantly, knockdown of pepck1, but not pepck2, within tumor cells not only diminishes the growth induced by HDS but also markedly enhances the survival rate of animals bearing Ras/Src tumors when they are fed HDS.Interestingly, overexpression of pepck1 or pepck2 did not further exacerbate tumor growth/developmental delay/lethality in animals bearing Ras/Src tumors under HDS.Furthermore, knockdown of pepck1 diminishes wingless and TOR signaling, decreases apoptosis and genome instability, and suppresses HDS-induced upregulation of glucose uptake and trehalose levels in tumor cells.Moreover, pharmacological inhibition of PEPCK1 using hydrazinium sulfate (HS) significantly enhances the survival of animals carrying Ras/Src tumors with knocked down expression of pepck1 under both NDS and HDS conditions.
Thus, our study reveals novel mechanisms by which aberrant upregulation of pepck1 promotes HDS-induced tumor progression, including enhanced wingless/Wnt and mTOR/ TOR signaling, apoptosis evasion, genome instability, and metabolic reprogramming with increased glucose uptake and trehalose levels.These findings provide greater understanding of the intricate relationship between diet and cancer and reveal potential targets for the prevention and treatment of cancers associated with metabolic disorders.

pepck1 is upregulated during HDS-induced tumor progression
Our prior research has shown that HDS decreases heterochromatin levels in tumor cells, and HP1a-mediated heterochromatin formation suppresses the tumor progression induced by HDS [18].These effects have raised questions about the specific downstream genes involved in the process of HDS-induced tumor progression through HP1a-mediated heterochromatin formation.By analyzing microarray databases from cultured Drosophila cells treated with HP1a RNAi and from Drosophila fed an HDS regimen [19][20][21], we identified several metabolic genes, including phosphoenolpyruvate carboxykinases1 (pepck1), as potential targets regulated by both HP1a and HDS (Supplementary Fig. S1A).To further explore how HP1a-mediated heterochromatin formation regulates metabolism, we utilized an RU486-inducible system for HP1a overexpression or knockdown in normal adult flies.Consistently, analysis revealed that levels of pepck1 were elevated in flies with HP1a knockdown and reduced in those with HP1a overexpression (Supplementary Fig. S1B, C).This suggests that HP1a-mediated heterochromatin formation plays a role in the downregulation of pepck1 in carbohydrate metabolism.This observation was further supported by chromatin immunoprecipitation (ChIP) assays using an H3K9me2 antibody, which demonstrated that heterochromatin is present in the pepck1 gene body (Supplementary Fig. S1D).Collectively, these findings suggest that heterochromatin formed through an HP1a-mediated process interacts directly with the pepck1 gene to downregulate pepck1 expression in vivo.
We then undertook a comprehensive investigation of transcriptional changes in glycolysis or gluconeogenesis genes following HDS-induced tumor progression.Experiments using tumor tissues from HDS-fed third instar larvae revealed increased expression of both cytosolic (pepck1) and mitochondrial (pepck2) forms of pepck mRNA, as well as elevated lactate dehydrogenase (Ldh) mRNA, indicative of the Warburg Effect in HDS-induced tumors (Supplementary Fig. S2A-E).
To investigate the role of PEPCK1 in HDS-induced tumor progression, we generated a pepck1 RNAi strain in Ras/Src tumor-bearing animals to specifically reduce pepck1 expression in tumors under HDS (Fig. 1).First, we assessed the efficacy of the transgenic pepck1 RNAi in flies, noted as pepck1 RNAi , and found a significant decrease in pepck1 mRNA levels in the Ras/Src tumor-bearing strain when it was combined with the pepck1 RNAi line (Fig. 1A; Supplementary Fig. S2B).Subsequently, we investigated pepck1 expression patterns in tumors using RNA in situ hybridization with digoxygenin (DIG)-labeled pepck1 antisense RNA probes in the eye discs of male ras G12V ; csk −/− and ras G12V , pepck1 RNAi ; csk −/− tumor-bearing animals under NDS or HDS conditions.The results revealed consistently increased pepck1 mRNA expression in HDS-induced tumor cells and validated efficient knockdown of pepck1 by RNAi in vivo (Fig. 1B, C).
Developmental delay and lethality are increased in tumorbearing animals exposed to HDS [18].Therefore, we investigated whether reducing pepck1 expression could reverse these defects in ras G12V , pepck1 RNAi ; csk −/− tumor-bearing animals.Remarkably, pepck1 knockdown was associated with significant reduction in developmental delay and increased survival rates of animals compared to the ras G12V ; csk −/− strain under HDS conditions (Fig. 1D-G).Next, we investigated whether the knockdown of pepck1 in tumors could alleviate tumor burden.Indeed, the tumor-bearing animals with pepck1 knockdown (ras G12V , pepck1 RNAi ; csk −/− ) showed notable reduction in GFP-positive tumor growth compared to tumor-bearing animals without pepck1 knockdown (ras G12V ; csk −/− ) (Fig. 1H-J).This finding suggests that PEPCK1 plays an important role in promoting the progression of HDS-induced tumors.Moreover, consistent with qPCR results (Fig. S2A-D), RNA in situ hybridization with an antisense pepck2 probe showed that pepck2 was upregulated during HDS-induced tumor progression, though to a lesser extent than pepck1 (Fig. S3A-C).However, the knockdown of pepck2 in tumor cells did not lead to significant changes in tumor growth, developmental delay, or lethality in Ras/Src tumor-bearing animals under HDS (Fig. S3D-H).Likewise, overexpression of pepck2 had no effect on tumor growth, developmental delay, or lethality in animals bearing Ras/Src tumors under HDS (Fig. S4A-F).Surprisingly, overexpression of pepck1 marginally decreased the developmental delay, (Fig. S5A-C) but did not exacerbate tumor growth, induced by HDS in animals (Fig. S5D, E), nor did it alter the lethality of Ras/Src tumor-bearing animals fed HDS (Fig. S5F).Together, these data indicate that aberrant expression of pepck1 in HDSinduced tumors contributes to developmental delay and reduced survival of animals, and knockdown of pepck1, but not pepck2, mitigates these effects, highlighting the essential role of pepck1 in HDS-induced tumorigenesis.

Knockdown of pepck1 reduces tumor burden and wingless signaling in HDS-induced tumors
The wingless/Wnt pathway, a highly conserved regulatory pathway, governs both normal cell development and tumor cell proliferation and progression [22][23][24][25].It has been observed that high dietary sugar (HDS) increases tumor cell insulin sensitivity and sugar influx, thereby promoting tumor progression through upregulation of wingless/Wnt signaling [6,18,26].To assess whether pepck1 knockdown reduces wingless/Wnt signaling in HDS-induced tumor growth, we utilized a specific antibody against wingless to evaluate wingless/Wnt signaling in tumor-bearing animals (ras G12V ; csk −/− ) and tumor-bearing animals with pepck1 knockdown (ras G12V , pepck1 RNAi ; csk −/− ) fed HDS or NDS.Compared to those without pepck1 knockdown, tumor-bearing animals with pepck1 knockdown exhibited a significant decrease in wingless protein expression within the tumor area during HDS-induced tumor growth (Fig. 2A-C).These results suggest that pepck1 knockdown reduces wingless signaling during HDS-induced tumor progression.Overall, our findings suggest that PEPCK1 contributes to HDS-induced tumor growth by upregulating wingless signaling.

Knockdown of pepck1 reduces trehalose levels and glucose uptake in HDS-induced tumor cells
In the adult Drosophila intestinal tumor model, tumorbearing animals exhibit high levels of trehalose, which is a predominant sugar in the hemolymph [27][28][29][30].Moreover, there is an increase in glucose uptake observed in Ras/ Src tumor cells under HDS conditions [6].Therefore, we next determined whether pepck1 regulates trehalose and glucose levels during HDS-induced tumor progression.We observed elevated levels of trehalose and glucose in Fig. 1 Reducing HDS-induced upregulation of pepck1 alleviates developmental delay and reduces lethality and tumor size among Ras/Src tumor-bearing animals.Drosophila with the following genotypes were used in these experiments: lacZ (control), ras G12V ; csk −/− (tumor-bearing), and ras G12V , pepck1 RNAi ; csk −/− (tumor-bearing with pepck1 knockdown).A Relative pepck1 mRNA levels were determined based on RNA extracted from 30 eye discs of combined 3rd instar male and female larvae (n = 15) fed HDS.Results were normalized to rpl32.B pepck1 mRNA expression patterns were examined using DIG-labeled pepck1 sense (negative control) or antisense RNA probes in WT male eye discs.C pepck1 mRNA expression patterns were analyzed using DIG-labeled pepck1 antisense RNA probes in the eye discs of male ras G12V ; csk −/− and ras G12V , pepck1 RNAi ; csk −/− Drosophila under NDS or HDS.D Pupation rates of combined male and female animals fed a 0.15 M sucrose diet (NDS) and a 0.75 M sucrose diet (HDS).E Number of days (PR 50 ) until pupation rate reached 50% among control and tumor-bearing animals.F, G Eclosion rates of combined male and female animals fed NDS or HDS.(H) Eye discs from 3rd instar ras G12V ; csk −/− and ras G12V , pepck1 RNAi ; csk −/− female Drosophila larvae fed NDS, with GFP-labeled tumor cells.I Eye discs from 3rd instar ras G12V ; csk −/− and ras G12V , pepck-1 RNAi ; csk −/− female Drosophila larvae fed HDS, with GFP-labeled tumor cells.J Percentage of GFP-positive tumor cells normalized to total eye disc area from female Drosophila fed NDS or HDS.Results are shown as mean ± SD.Asterisks indicate statistically significant differences via two-way ANOVA with paired controls (*P < 0.05, **P < 0.01).GFP green fluorescent protein, HDS high dietary sugar, NDS normal dietary sugar, N.S. not significant; SD standard deviation the HDS-induced tumor group and showed that reduction of pepck1 in the tumor reversed the HDS-mediated increase in trehalose levels without affecting glucose levels (Fig. 3A, C).Moreover, trehalose-6-phosphate phosphatase (T6Pase, TPS2) mRNA, which encodes an enzyme that converts trehalose-6-phosphate into trehalose, was upregulated in HDS-induced tumors; importantly, knockdown of pepck1 in Ras/Src tumors under both NDS and HDS conditions resulted in significant reduction of T6Pase levels (Fig. 3B).These results suggest that pepck1 not only maintain trehalose homeostasis under NDS but also mediates HDS-induced elevation of trehalose levels in tumor cells through T6Pase.To further investigate whether pepck1 regulates glucose uptake, we measured uptake rates of Ras/Src tumor tissue exposed to the glucose analog 2NBDG for a controlled period of time.HDSinduced tumors exhibited consistently elevated glucose uptake, and notably, reduction of pepck1 levels decreased glucose uptake (Fig. 3D-H).Overall, these results suggest that the upregulation of PEPCK1 by HDS increases trehalose levels and glucose uptake, thereby enhancing glucose metabolism in tumor cells.

Knockdown of pepck1 reduces mTOR/TOR signaling during HDS-induced tumor progression
mTOR/TOR signaling, a crucial nutrient-sensing pathway, is upregulated in the progression of multiple types of cancer [31][32][33][34][35]. Previous work specifically demonstrated that TOR-S6K signaling is activated in Ras/Src tumors under a 1.0 M sucrose HDS diet, as assessed by phosho RpS6 (pS6) antibody staining [5].To determine whether upregulated PEPCK1 enhances mTOR/TOR signaling and thus promotes tumor growth under HDS conditions, we assessed mTOR/TOR signaling activity using pS6 antibody, a specific antibody against phosphorylated Ribosomal protein S6 (RpS6) [36], in tumor cells.We observed reduction in pS6 staining intensity in the tumor areas in animals with pepck1 knockdown after day 8 of HDS-induced tumor growth.Under NDS conditions, pS6 staining intensity in tumor tissue from animals with pepck1 knockdown showed no significant difference compared to tissue from control tumor-bearing animals (Fig. 4A-C).We found that pepck1 knockdown consistently resulted in significant reduction in the expression of TOR, 4EBP1, and S6 genes in tumor-bearing animals under HDS conditions, but not under NDS conditions (Fig. 4D-F).Therefore, pepck1 knockdown suppressed TOR signaling associated with progression of HDS-induced tumors.These findings collectively suggest that PEPCK1 promotes mTOR/TOR signaling during HDSinduced tumor progression.[6,18].Therefore, we investigated whether upregulated pepck1 Fig. 3 Knockdown of pepck1 reduces HDS-induced elevated trehalose levels and glucose uptake in tumor cells.A Trehalose levels were measured using 60 eye discs from combined 3rd instar ras G12V ; csk −/− and ras G12V , pepck1 RNAi ; csk −/− male and female Drosophila larvae fed NDS or HDS.B Relative levels of TPS2 (trehalose-6-phosphate phosphatase) mRNA were determined based on RNA extracted from 30 eye discs of combined 3rd instar ras G12V ; csk −/− and ras G12V , pepck1 RNAi ; csk −/− male and female Drosophila larvae fed NDS or HDS.C Glucose levels were assessed using 60 eye discs from combined 3rd instar ras G12V ; csk −/− and ras G12V , pepck1 RNAi ; csk −/− male and female Drosophila larvae fed NDS or HDS.Results are shown as mean ± SD.Asterisk indicates a statistically significant difference via two-way ANOVA with paired control (*P < 0.05; ***P < 0.001).D, E 2NBDG expression (red) in eye discs from 3rd instar ras G12V ; csk −/− and ras G12V , pepck1 RNAi ; csk −/− female Drosophila larvae fed NDS, with GFP-labeled tumor cells (green).F, G Expression of 2NBDG (red) in eye discs from 3rd instar ras G12V ; csk −/− and ras G12V , pepck1 RNAi ; csk −/− female Drosophila larvae fed HDS, with GFP-labeled tumor cells (green).H Quantification of 2NBDG levels (red) via fluorescence intensity in tumor cells (green) in eye discs from 3rd instar ras G12V ; csk −/− and ras G12V , pepck1 RNAi ; csk −/− female Drosophila larvae fed NDS or HDS.Fluorescence intensity was quantified using the ZEISS ZEN Blue software.Results are shown as mean ± SD.Asterisks indicate statistically significant differences via two-way ANOVA with paired control (*P < 0.05).GFP green fluorescent protein, HDS high dietary sugar, NDS normal dietary sugar, N.S. not significant, SD standard deviation decreases apoptosis in HDS-induced tumor cells.Using the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, which identifies cells undergoing programmed cell death by detecting DNA fragmentation, we observed a similar number of TUNEL-positive foci in tumors under both NDS and HDS conditions, which is consistent with previous findings from our group [18] (Fig. 5A-C).Moreover, pepck1 knockdown in HDS-induced Ras/Src tumors significantly increased apoptosis in tumor cells (Fig. 5A-C).These results indicate that the anti-apoptotic effect observed in HDS-induced tumors can be reversed by downregulation of pepck1.These findings suggest that HDS-induced upregulation of PEPCK1 contributes to tumor cell evasion of apoptosis.

Knockdown of pepck1 reduces HDS-induced genome instability in tumor cells
Genome instability significantly contributes to the development of tumors [8].Moreover, genome instability plays a crucial role in the development and progression of tumors in Drosophila [37].Notably, a previous report investigated Fig. 4 Knockdown of pepck1 decreases TOR signaling in tumor cells under NDS and HDS.Eye discs from ras G12V ; csk −/− and ras G12V , pepck1 RNAi ; csk −/− 3rd instar larvae, fed NDS or HDS, with GFPlabeled tumor cells (green) and pS6 immunostaining (red).Scale bar: 100 µm.A ras G12V ; csk −/− and ras G12V , pepck1 RNAi ; csk −/− female larvae fed NDS.B ras G12V ; csk −/− and ras G12V , pepck1 RNAi ; csk −/− female larvae fed HDS on day 7 AEL.C Quantification of pS6 fluorescence intensity in tumor cells from female larvae fed HDS; n = 9 eye discs per group.Fluorescence intensity was quantified using the ZEISS ZEN Blue software.Results are shown as mean ± SD of individual eye discs.Differences between groups were assessed via two-way ANOVA; *P < 0.05.Relative levels of TOR (D), 4EBP1 (E) and S6 (F) mRNA were determined based on RNA extracted from 50 eye discs from combined 3rd instar ras G12V ; csk −/− and ras G12V , pepck-1 RNAi ; csk −/− male and female Drosophila larvae fed NDS or HDS.Results are shown as mean ± SEM.Asterisks indicate statistically significant differences via Student's t-test (*P < 0.05).AEL after egg laying, GFP green fluorescent protein, HDS high dietary sugar, NDS normal dietary sugar, SD standard deviation the link between pepck1 and chromosomal instability in a Drosophila brat tumor explant model [38].Therefore, we investigated whether upregulation of pepck1 promotes HDSinduced genome instability in tumor cells.Consistent with our previous findings [18], the number of γ-H2AX foci, a marker for double-stranded DNA breaks and DNA damage, was increased in tumor cells under HDS conditions.Importantly, pepck1 knockdown reduced the number of γ-H2AX foci in HDS-induced tumor cells (Fig. 6A-C).Therefore, pepck1 knockdown enhances genome stability in tumor cells under HDS, suggesting that pepck1 increases genome instability in HDS-induced tumor cells.

Hydrazinium sulfate further decreases HDS-induced developmental delay and increases survival of tumor-bearing animals with pepck1 knockdown
To assess whether pharmacological targeting of PEPCK1 offers a preventive and therapeutic strategy against HDSinduced tumorigenesis in vivo, we employed hydrazinium sulfate (HS), an inhibitor of PEPCK, in an attempt to suppress tumor progression by inhibiting PEPCK1 [38].The mechanism of action for HS involves blocking the binding site of the PEPCK enzyme on oxaloacetate (OAA), thereby impeding the conversion of OAA to phosphoenolpyruvate (PEP) [39].Additionally, HS reduces gluconeogenesis by inhibiting PEPCK, which decreases both pyruvate carboxylase (PC) activity and the transport of pyruvate to the mitochondria [40].Initially, we investigated whether inhibiting PEPCK1 could reduce developmental delay and lethality induced by HDS in tumor-bearing animals.We evaluated the eclosion rates of wild-type (lacZ), tumor-bearing animals (ras G12V ; csk −/− ), and tumor-bearing animals with pepck1 knockdown (ras G12V , pepck1 RNAi ; csk −/− ), respectively, that were treated with HS at concentrations of 0 µM, 10 µM, 50 µM, and 100 µM.In our study of survival rates of tumor-bearing animals, we observed that HS is non-toxic to wild-type (lacZ) Drosophila, regardless of whether they were fed NDS or HDS (Fig. 7A, B).Additionally, we observed a significant increase in the survival rate of HDS-fed tumor-bearing animals with pepck1 knockdown (ras G12V , pepck1 RNAi ; csk −/− ) treated with 10 µM HS, compared to those receiving no HS (0 µM treatment) (Fig. 7).Moreover, under NDS, the survival rates of tumor-bearing animals (ras G12V ; csk −/− ) decreased with 50 µM HS treatment compared to those not treated with HS (0 µM treatment) (Fig. 7).In contrast, the survival rate of NDS-fed tumor-bearing animals with pepck1 knockdown (ras G12V , pepck1 RNAi ; csk −/− ) improved with a 10 µM HS treatment (Fig. 7).Thus, the combined treatment with 10 µM HS and pepck1 knockdown significantly reduces the lethality of tumor-bearing animals.Overall, these results indicate that inhibition of PEPCK by HS effectively enhances the survival of tumor-bearing animals with pepck1 knockdown under conditions of HDS.

Discussion
Globally, more than 650 million adults are clinically obese, with excessive consumption of added sugars significantly contributing to metabolic disorders and associated cancers [41,42].Better understanding of the complex relationship between diet and cancer at molecular, cellular, and organismal levels, by exploring the role of metabolic alterations Fig. 6 Knockdown of pepck1 reduces HDS-induced DNA damage in tumor cells.A, B Eye discs from ras G12V ; csk −/− and ras G12V , pepck1 RNAi ; csk −/− 3rd instar female Drosophila larvae, fed NDS or HDS, with GFP-labeled tumor cells (green) and γH2AX immunostaining (white).Scale bar: 100 μm.C Quantification of γH2AX foci in tumor cells; n = 6 female eye discs per group.Results are shown as mean ± SD.Asterisks indicate statistically significant differences via two-way ANOVA with paired control (*P < 0.05; **P < 0.01).GFP green fluorescent protein, HDS high dietary sugar, NDS normal dietary sugar, N.S. not significant, SD standard deviation in promoting HDS-induced cancer progression, is crucial for the prevention and treatment of diet-associated cancers.In this study, we employed a range of techniques, including bioinformatics, and discovered that pepck1 is surprisingly upregulated by HDS during tumor progression.Interestingly, pepck1 is directly downregulated by HP1a-mediated heterochromatin formation in wild-type adult flies.Our previous studies have consistently shown that HP1a-mediated heterochromatin formation is a significant epigenetic factor in suppressing tumor development induced by a high sugar diet, while HDS itself downregulates HP1a during tumor progression [18].Therefore, HDS-induced reduction of HP1a-mediated heterochromatin formation may explain aberrant epigenetic upregulation of pepck1 during tumor progression induced by a diet that is high in sugar [18].However, it remains unclear whether HDSmediated increase in pepck1 gene expression in tumors is due to direct epigenetic modulation by HP1a-mediated heterochromatin formation.To address this in future studies, HP1a-mediated heterochromatin formation should be promoted using the ras G12V , HP1a; csk −/− fly line [18] and tested to determine whether the pepck1 mRNA levels are decreased in ras G12V , HP1a; csk −/− tumors under HDS.Furthermore, to demonstrate that increased pepck1 is due to epigenetic changes, a ChIP assay of the pepck1 gene from Ras/Src-tumors under NDS and HDS should be performed.Moreover, PEPCK1, a crucial cytosolic enzyme in gluconeogenesis, is upregulated in many human cancers, indicating its significant role in cancer progression [15].However, to our knowledge, this study is the first to identify the paradoxical upregulation of PEPCK1 by HDS during tumor progression.Thus, we aimed to determine the role of PEPCK1 in HDS-induced cancer progression by utilizing the Drosophila Ras/Src cancer model, which shares many hallmarks of human cancer progression [43].Our research uncovered new mechanisms through which the abnormal enhancement of PEPCK1 accelerates tumor growth in response to a high sugar diet.This involves the activation of the wingless/Wnt and mTOR/TOR signaling pathways, suppression of apoptosis, increased genome instability, and the reprogramming of carbohydrate metabolism to result in increased glucose uptake and trehalose levels.These insights deepen our understanding of the complex link between diet and cancer, highlighting potential points of intervention for prevention and treatment of cancers linked to metabolic disorders.Numerous cancer types exhibit alterations in key enzymes involved in gluconeogenesis that contribute to reprogramming of metabolic pathways or induce tumor progression [44][45][46].These adjustments increase metabolic flexibility and permit the incorporation of non-carbohydrate substances in biosynthesis and the modification of glucose flows to enhance antioxidant production.Specifically, PEPCK1 promotes colon cancer growth by increasing gluconeogenesis metabolites through phosphoenolpyruvate and pyruvate production [14].Likewise, pepck2 gene expression is increased in thyroid, bladder, breast, kidney, and non-small-cell lung cancer [44].Interestingly, prior research has suggested that PEPCK1 plays tumor suppressor role in liver cancer [47,48].These findings highlight the complex roles of PEPCK1 and PEPCK2 in cancer progression across various types of human cancer, despite the tumor-suppressive function of PEPCK1 in hepatocellular carcinoma.Our research reveals that enzymes such as pepck1, pepck2, and Ldh are overexpressed during tumor progression, after being triggered by HDS.Furthermore, aberrant upregulation of pepck1 leads to elevated trehalose synthesis and glucose uptake.Thus, we have uncovered new mechanisms by which PEPCK1 facilitates reprogramming of carbohydrate metabolism during HDS-induced tumor progression.
In this study, we discovered that knockdown of pepck1, but not pepck2, reduces tumor progression induced by HDS.We consistently found that overexpression of pepck2 does not enhance tumorigenesis under HDS conditions.Interestingly, even overexpression of pepck1 does not increase tumor progression under NDS or HDS.These results suggest that the maximum potential of PEPCK1 to promote tumor growth may already be reached under HDS, resulting in no additional effect of overexpression.In other words, HDS conditions likely maximize pepck1 expression and its associated effects in promoting tumor growth in the Ras/ Src cancer model.Importantly, PEPCK1 does not appear to influence tumor progression under NDS, highlighting its role as specific to the HDS context.Conversely, PEPCK2 does not significantly affect tumor progression, in either promotion or inhibition, or under either HDS or NDS conditions, indicating a possible specificity in the function of PEPCK1 and PEPCK2 related to cancer type and/or dietary context combination.
In this study, we demonstrated that the knockdown of pepck1 in Ras/Src tumor cells suppresses HDS-induced genome instability (Fig. 6).Notably, the HDS-induced increase in γ-H2AX foci and the genome instability suppression effect from pepck1 knockdown are observed across the entire tissue, not just in the tumor cells.This finding aligns with our previous observation of non-cell-autonomous DNA damage occurring in non-tumor cells adjacent to tumor cells [18].Furthermore, a similar non-cell-autonomous effect was observed concerning HDS-induced evasion of apoptosis and the influence of pepck1 knockdown within tumor cells (Fig. 5) [18].The molecular mechanisms underlying these non-cell-autonomous effects are not yet fully understood and are currently under investigation.In Drosophila, apoptotic cells have been shown to produce non-cell-autonomous signals, including wingless, Dpp, and TNF, which can induce compensatory apoptosis in neighboring cells within the wing imaginal disc [49].Additionally, chromosomal instability has been implicated in promoting non-cell-autonomous cancer progression through interactions with the immune system in both mouse cancer models and human breast cancer cells [50].Future studies are needed to elucidate the mechanisms driving non-cell-autonomous apoptosis and genome instability effects on HDS-induced tumor progression.
Here, we found that targeted pharmacological inhibition of PEPCK1 with HS significantly enhances the survival of tumor-bearing animals with pepck1 knockdown under NDS and HDS.Our findings align with a previous study that also demonstrated the effectiveness of HS in inhibiting PEPCK1 and suppressing brain tumor growth in Drosophila [38].Interestingly, the survival of ras G12V , pepck1 RNAi ; csk −/− animals is increased compared to ras G12V ; csk −/− animals under HDS (Fig. 1G), while HS treatment did not affect the survival of ras G12V ; csk −/− animals under HDS (Fig. 7B; middle panel).It is likely that the concentrations of 10 µM, 50 µM, and 100 µM HS may not be high enough to inhibit the upregulated levels of PEPCK1 in ras G12V ; csk −/− animals under HDS.Future studies are necessary to test whether feeding HS alone at a higher concentration, such as 10 mM of HS mixed in the fly diet [38], is sufficient to enhance the survival of ras G12V ; csk −/− animals.Moreover, combining 10 µM HS treatment with pepck1 knockdown significantly decreases the lethality in tumor-bearing animals under both NDS and HDS (Fig. 7A, B; right panels).It is likely that 10 µM HS treatment only improves survival in animals with ras G12V , pepck1 RNAi ; csk −/− tumors due to the effective reduction of PEPCK1 levels by pepck1 RNAi, which allows 10 µM HS to sufficiently inhibit PEPCK1 in this situation.Furthermore, future studies should determine whether the effect of HS on the increased survival of ras G12V , pepck1 RNAi ; csk −/− animals is due to tumor suppression rather than systemic effects independent of tumor growth by measuring the tumor size in Fig. 7.
Overall, our research adds to the understanding of the molecular, cellular, and organismal mechanisms through which upregulation of PEPCK1 leads to HDS-aggravated tumor progression.These findings deepen our understanding of the intricate connections between diet and cancer through cellular and molecular processes, revealing new potential targets for both preventing and treating cancers linked to metabolic disorders, as well as mitigating the impact of dietary factors on cancer progression (Fig. 8).

Fly cultures, immunofluorescence, quantitative RT-PCR, RNA in situ hybridization, Glucose and trehalose assay, ChIP assay and TUNEL assay
The detailed procedure is described in Supplementary Materials and Methods.

Geneswitch inducible system
To activate specific gene expression, adult flies were maintained on a diet that included food mixed with 300 μM RU486 or with solvent (ethanol) only as a control.Following four days of feeding, the flies were homogenized and their cells were lysed to extract RNA.

Statistical analysis
The data presented in this study were derived from a minimum of three separate experiments and were analyzed using PRISM 6 software (GraphPad, San Diego, CA).The results were expressed as the mean ± standard deviation (SD).Statistical significance was assessed using two-way Fig. 8 High sugar diet induces upregulation of pepck1, which promotes cancer progression by acivating wingless/dWnt and TOR signaling, decreasing apoptosis, inducing genome instability, and reprogramming carbohydrate metabolism.In the presence of HDS, tumor cells upregulate PEPCK1 to increase tumor burden and decrease the survival rate of tumor-bearing animals.Additionally, the heterochromatin formed via an HP1a-mediated process directly suppresses pepck1 expression in tumor cells.Mechanistically, PEPCK1 facilitates HDSinduced tumor progression by elevating wingless expression and TOR signaling, suppressing apoptosis, inducing genome instability, and reprogramming carbohydrate metabolism through increased glucose uptake and elevated trehalose levels ANOVA or Student's t-test, as indicated in the respective figure legends.A P-value of less than 0.05 was considered statistically significant.

Fig. 7
Fig.7 Hydrazinium sulfate (HS), an inhibitor of PEPCK, enhances the survival of tumor-bearing animals with pepck1 knockdown.Drosophila larvae with the following genotypes were fed either NDS or HDS and used in the experiments: lacZ, and ras G12V ; csk −/− and ras G12V ; pepck1 RNAi ; csk −/− .A total of 200 combined male and female larvae per genotype were used for each experiment.A Eclosion rates of animals fed NDS with treatments of 0 µM, 10 µM, 50 µM,